An elevation in the [Ca2+], following secretagogue stimulation plays a fundamental role in underlying digestive enzyme secretion from the exocrine pancreas. The increase in [Ca2+] is a tightly regulated event and exhibits characteristic temporal and spatial patterning which is absolutely required for appropriate exocrine function. Of note, disruption of Ca2+ signaling occurs as an early event in models of pancreatitis. A major determinant of the characteristics of these Ca2* signals is through the properties of inositol 1,4,5- trisphosphate receptors (lnsP3R). This is best illustrated by a recent study reporting that ablation in mice of the type-2 and type-3 lnsP3R resulted in death because of failure of this signaling pathway and thus of digestive enzyme secretion. In stark contrast to the lnsP3R-1, little information is available regarding the specific biophysical and electrophysiological properties of lnsP3R-2/3- obviously crucial to pancreatic exocrine function. We will therefore define the fundamental properties of these receptors and thus investigate their individual contributions to acinar cell signaling. The specific aims that will be addressed will investigate the regulation of lnsP3R-2 and lnsP3R-3 by Ca2+ (aim 1) by intracellular ATP (aim 2) and following PKA phosphorylation (aim 3). The biophysical data will be integrated into mathematical models of lnsP3R and exocrine function. The single-channel electrophysiological properties of each receptor will be studied using whole-cell patch-clamp by exploiting the expression of lnsP3R in the plasma membrane of a series of stable cell lines expressing lnsP3R-2 or lnsP3R-3 and mutant constructs in isolation. These data will be complemented by study of the properties of native lnsP3R expressed in the ER membrane by "on-nucleus" patching of preparations of isolated pancreatic nuclei from wild-type or lnsP3R-2 null animals. Ca2+ release will be monitored using a unidirectional flux assay in permeabilized cells and by digital imaging in intact cells. The proposal will provide a detailed understanding of the fundamental processes controlling Ca2+ signaling and thus normal function of the exocrine pancreas. The long term goal of the studies is driven by the central idea that a detailed knowledge of normal function is absolutely necessary to understanding how these mechanisms are disrupted in pancreatic disease states and thus for the ultimate development of novel strategies for the treatment of disease.